The present invention relates to a method for culturing algae having an ability of producing highly unsaturated fatty acids, photosynthetic pigments, and/or polysaccharides to manufacture the algae containing these substances in algal cells, more specifically relates to the method for culturing algae having an ability of producing the highly unsaturated fatty acids, the photosynthetic pigments, and/or the polysaccharides in a culture medium under light irradiation and/or aeration condition to manufacture efficiently algae containing these highly unsaturated fatty acids, photosynthetic pigments, and/or polysaccharides in algal cells in a high concentration using a certain cultivation apparatus.
Development of a cultivation method for algae, wildly growing, is needed for stable, high yield production of the photosynthetic pigment used as a safe feed for a cultured fish and as a safe food additive and unsaturated fatty acid, photosynthetic pigment, and/or polysaccharide used for a medical drug and a health food. However, cultivation method has been limited to a few numbers and thus, very limited species such as Chlorella and Spirulina were studied and a method for an efficient cultivation is has not been yet established.
Algae perform biosynthesis of a useful substance by photosynthesis through absorption of carbon dioxide. In addition, it is important that algal cultivation is carried out efficiently and therefore, an apparatus for cultivation is necessary for efficient photosynthesis. Hence, improving the apparatus for cultivation and developing a new apparatus for cultivation are being conducted.
So far, as the apparatus for cultivation for algae, for example, a cultivation pond, a raceway type cultivation apparatus, a tubular type cultivation apparatus, a liquid membrane-forming cultivation apparatus have been well known. In the cultivation pond, a concrete-made cultivation pond or cultivation vessel are prepared in the outdoors to build up a pond in which a culture solution is filled and a species of a microalgae exemplified by Chlorella is cultured in the pond using sunlight. However, this type of the cultivation apparatus requires such area as 3000 m2 for the pond resulting in a huge volume.
In addition, cultivation of microalgae by using the cultivation apparatus of this type causes a high concentration of algal cells containing in the culture solution according to progress of cultivation resulting in thick green coloration of the culture solution to disturb reaching of sunlight to a bottom of the pond. From such phenomenon, the following problem comes up: unless algal cell density necessary for cultivation is reduced, a total algal photosynthetic efficiency is lowered.
Consequently, a depth of the pond must be set to 15 cm or smaller and a land for mass cultivation of algal cells must be vast. Further, a concentration of the culture solution cannot be made higher and thus, in collection of a culture from the solution, the following problem comes up: the culture must be collected from a huge amount of a low density culture solution.
On the other hand, the cultivation pond must be stirred to make algal photosynthesis easy. However, a large amount of energy is required for stirring the huge volume of the low concentration liquid. In addition, the cultivation pond is located in the outdoors and in an opened state and hence, such exogenous matters as dust and waste are easy to contaminate the liquid and airborne microorganisms and cells of other species of microalgae contaminate the pond to propagate resulting in the following problem: culture of a high purity and a high quality cannot be yielded.
The cultivation pond is located in the outdoors and thus, a temperature thereof changes according to a change of weather to cause very difficulty to maintain the temperature of the pond in a constant value. Particularly, there is a defect that in winter in a certain locality, the temperature drops excessively.
Caused by such status, cultivation of algae by using the cultivation pond cannot be applicable to other algal genera than Chlorella, Spirulina, and Dunaliella, which can grow under a unique condition such as a high pH and a high salinity.
The raceway type cultivation apparatus has a circulatory path, for culture solution, made by partitioning of an inside of the cultivation vessel with a straightening vane and is used for cultivation of algae by the method by which the culture solution is circulated in this circulatory path by using means for circulation. This system is of an improved circulation system of the culture solution and a photosynthetic rate of algae lowers, similar to the cultivation method using the cultivation pond, according to progress of cultivation and therefore, does not allow efficient use of light energy. Consequently, the following problem also comes up: an efficiency of carbon dioxide is low. In order to achieve efficient use of light, irradiation of sunlight to the liquid through an optical fiber has been proposed (Japanese Utility Model Application Laid-Open No. 5-43900).
In operating algal cultivation by using this system, the liquid is circulated by mechanical stirring and thus, the following inevitable defects occur: algal cells are broken and a shear stress occurs (a phenomenon in which algal cells are cut by a shear stress to cause reduction of cellular activity resulting in a low propagation rate).
The tubular type cultivation apparatus is the apparatus for algal cultivation by using the cultivation apparatus comprising a light transmission tube. Algal cultivation by using this apparatus allows no contamination of the culture solution by non-objective bacteria and making culture concentration high and thus, is a very beneficial method for collection of the useful substance, which is produced by algae, by separating algae from the culture solution.
However, after long-time algal cultivation, algae attach to an internal wall of the tube to disturb light pass through the tube very. These facts make efficient algal cultivation difficult and also make removal of algae attached to the internal wall of the tube difficult.
In order to solve these problems, the following method has been proposed: a cleaning ball is put in the tube an the ball is always circulated together with the culture solution to clean the internal wall of the tube (Japanese Patent Application Laid-Open No. 6-90739). However, even by this method, the following and other many problems remain: dirt and algal attachment of the internal wall of the tube cannot be continuously removed completely, the ball must be collected to clean and also, the ball must be always circulated in the tube. In addition, a further problem of this system for algal cultivation is that cultivation operated inside the tube allows oxygen gas, which is generated by algal photosynthesis to stay, in the tube and oxygen inhibits algal photosynthesis. Based on these facts, a device of the apparatus has been proposed to suppress a bad effect caused by oxygen, generated by photosynthesis, on cultivation (Japanese Patent Application Laid-Open No. 9-121835).
In the liquid membrane-forming cultivation apparatus, a dome-shaped, light-passing lid body is installed on an top face of the cultivation vessel and culture solution is jetted from a bottom to an internal face of an apical part of the dome-shaped lid body to form a liquid membrane on the internal face of the lid body and irradiate a light on this liquid membrane (Japanese Patent Application Laid-Open No. 8-38159).
However, there are problems: this system proposed requires a circulation pump for continuation of formation of the liquid membrane, is not suitable for mass cultivation, and does not allow using sunlight.
A panel type cultivation apparatus is used for algal cultivation by installing a thin box-like apparatus, which is prepared by using two resin-made panel boards, with inclination.
This apparatus itself is a closed type similar to the tubular type cultivation apparatus, for example, and has an advantage of no contamination of the culture solution by bacteria and wastes. However, the following problem come up: oxygen gas generated according to progress of cultivation dissolves in the culture solution and stays in the apparatus to inhibit algal photosynthesis.
In addition, in the case where the apparatus is installed in the outside, an altitude of the sun and a sunlight incident angle to a surface of the apparatus change according to time from sunrise to sunset to cause an insufficient total amount of sunlight received per an area, where it has been installed.
Japanese Patent Application Laid-Open No. 10-304867 discloses the method for designing conveniently the panel type cultivation apparatus having a most suitable light environment. In the disclosure, by combination of a relation between a passed light amount and a matter production activity with the relation between the passed light amount and the light path length of a reactor, the method for designing conveniently the light path length of the cultivation apparatus was created and the cultivation apparatus designed by this method was proposed.
However, the above described principal problems have not been solved.
Algae accumulate useful substances in their bodies by photosynthesis and the most important subject is efficient algal photosynthesis as possible. A factor for efficient photosynthesis may be increase in a light-receiving area of the cultivation apparatus, efficient stirring of the culture solution, adjusting a thickness or a depth of the culture solution, easy removal and cleaning of algal cells attached to the internal surface of the cultivation apparatus, temperature regulation, prevention of contamination of bacteria, cells of other algal species, and waste.
The problem of the light-receiving area is influenced by the larger light-receiving area as possible and efficient irradiation of the light on the culture solution.
For example, in the cultivation vessel and the cultivation pond, the surface area thereof is determined by the surfaces of the cultivation vessel and the cultivation pond and hence, in order to increase the surface area, increase in a size of the vessel and pond is an only one way to achieve it and there is no other way.
Stirring the culture solution is essential for even irradiation of the light on the cultivation pond and normally, means thereof are frequently by stirring or moving the liquid by using the pump and mechanical stirring in the vessel and pond.
However, such mechanical stirring causes break of and the shear stress to algal cells resulting in bad effects.
The photosynthesis rate differs between algal species and therefore, for species having a low rate and fast rate, the depth of the culture solution must be changed and the depth must be changed according to the objective culture concentration. As described above, the thickness or depth of the culture solution must be freely adjusted according to such conditions as algal species and the objective culture concentration.
Removal and cleaning of algae attached to the internal surface of the cultivation apparatus can be normally carried out in the outdoor open type cultivation pond and cultivation vessel. However, In the closed type cultivation apparatus, attached algae do not allow the light to pass and thus, removal and cleaning of algae must be carried out. Subsequently, at a stage of completion of cultivation, for operation of the next cultivation, the apparatus must be adapted to have a structure by which the internal surface of the cultivation apparatus must be cleaned and attached matter must be readily removed.
Temperature regulation is very important particularly for the closed apparatus to prevent occurrence of cultivation trouble caused by excessive rise of the temperature of the liquid in summer. In order to solve-this problem, the following method is known: cool water is mixed into the culture solution. However, the culture solution is diluted and hence, a huge amount of the culture solution diluted is treated in collection of the culture in the next step. Therefore, this method is very disadvantage for industrial application.
The cultivation apparatus is the apparatus used normally in the outdoors or the apparatus used normally in doors. By this reason, the following problem occurs: when the apparatus for the outdoors is used in doors, efficiency of light use becomes low and on the other hand, another problem occurs: the apparatus for use in doors is not used in the outdoors. The cultivation apparatus with a simple structure is desired to operate cultivation under normal condition cultivation in doors and in the outdoors.
Stirring the culture solution is an essentially necessary operation for even cultivation.
This is because of the following reasons: 1) a difference occurs between cultivation rates of a surface layer part and a deep layer part of the liquid medium, 2) such gas as air and carbon dioxide must be evenly distributed in the liquid medium, in other words, a whole of the culture solution, 3) the light must be evenly distributed in algae for cultivation, 4) prevention of sedimentation of algae readily making a colony during cultivation to stay in the bottom of the liquid and dispersion repeated in the culture solution.
It is required to stir always such culture solution and supply air or carbon dioxide gas to the culture solution. However, such conventional cultivation method has various issues as described above and therefore, is not a sufficient cultivation method.
The present invention improves defects of the conventional cultivation method as described above and has objects to provide the method for algal cultivation in the high yield and at a low cost to produce the photosynthetic pigment used as the safe feed for the cultured fish and ads the safe food additive and the unsaturated fatty acid, or polysaccharide used for a medical drug and the health food and the method for manufacturing algae containing the unsaturated fatty acid, photosynthetic pigment and/or polysaccharide in algal cells in a high concentration efficiently and in a state without contamination by impurities,
In order to solve the above described subjects, according to the present invention, the culture medium is filled in a gap and a space made by an internal clear material and an external clear material, air or carbon dioxide gas is injected from the bottom part and the light is irradiated under an aeration condition to make algae photosynthesize for production of the unsaturated fatty acid, photosynthetic pigment and/or polysaccharide in algal cells in a high quantity to manufacture algae containing these substances. Through such steps, high concentration cultivation can be achieved without contamination by algal cells of other species, waste, and bacteria.
The present invention is the method for culturing algae, which has the ability of producing the highly unsaturated fatty acid, the photosynthetic pigment, and/or the polysaccharide, in the high concentration in the culture medium by using the cultivation apparatus under light irradiation and aeration conditions to manufacture the algae containing the highly unsaturated fatty acid, the photosynthetic pigment, and/or the polysaccharide, wherein the above described cultivation apparatus employed is that selected from those with any one shape of the dome shape, conical shape, or cylindrical shape;
the cultivation apparatus with the dome shape comprises an external hemispheric dome made from a clear material, an internal hemispheric dome made from a clear material, and the bottom part connecting bottom end parts of both the domes, and a cylindrical opening member is installed on a top part of the external hemispheric dome and a member for leading air and/or carbon dioxide gas and a discharging member for the culture solution are installed on the bottom part;
the cultivation apparatus with the conical shape comprises an external conical circumferential wall made of the clear material, a clear internal conical circumferential wall, and the bottom part connecting bottom end parts of both the circumferential wall, and a cylindrical opening member is installed on the top part of the external conical circumferential wall and a member for leading air and/or carbon dioxide gas and a discharging member for the culture solution are installed on the bottom part;
or, the cultivation apparatus with the cylindrical shape comprises an external cylindrical circumferential wall made of the clear material and having an upper wall an internal cylindrical circumferential wall having the upper wall made of the clear material, and the bottom part connecting bottom end parts of both the circumferential wall, and the cylindrical opening member is installed on a central part of the upper wall of the external cylindrical circumferential wall and a member for leading air and/or carbon dioxide gas and a discharging member for the culture solution are installed on the bottom part.
In addition, the method for culturing algae, which has the ability of producing the highly unsaturated fatty acid, the photosynthetic pigment, and/or the polysaccharide, in the high concentration in the culture medium by using the cultivation apparatus under light irradiation and aeration conditions to manufacture the algae containing the highly unsaturated fatty acid, the photosynthetic pigment and/or the polysaccharide, wherein the above described cultivation apparatus used is the apparatus comprises a main body of the cultivation apparatus and a gas discharging apparatus; and
the main body of the cultivation apparatus is the cultivation apparatus with the dome shape, conical shape, or cylindrical shape, wherein
the cultivation apparatus with the dome shape comprises the external hemispheric dome made from a clear material, the internal hemispheric dome made from the clear material, and the bottom part connecting bottom end parts of both the domes, and a cylindrical opening member is installed on the top part of the external hemispheric dome and a discharging member for the culture solution is installed on the bottom part;
the cultivation apparatus with the conical shape comprises the external conical circumferential wall made of the clear material, the internal conical circumferential wall made from the cleat material, and the bottom part connecting bottom end parts of both the circumferential wall, and a cylindrical opening member is installed on the top part of the external conical circumferential wall and a discharging member for the culture solution is installed on the bottom part;
or, the cultivation apparatus with the cylindrical shape comprises the external cylindrical circumferential wall having the upper wall made of the clear material, the internal cylindrical circumferential wall having the upper wall made of the clear material, and the bottom part connecting bottom end parts of both the circumferential wall, and a cylindrical opening member is installed on the central part of the upper wall of the external cylindrical circumferential wall and a discharging member for the culture solution is installed on the bottom part;
the gas discharging apparatus is configured by opposite two square base board, a bubble leading member, of which section has a square shape lacking a side or a reversed U-shape, opened downward, and a discharge nozzle, the bubble leading member is installed with inclination to an upper side face of the square base board, forms the upper wall made by extending an inclined wall of the upper face of the bubble leading member by bending almost horizontally in the upper end part, and has a side wall extending from both the side ends of the inclined wall and the upper wall, and each of the bottom end parts of both the side walls are jointed on the two upper side faces of the square base board, and the discharge nozzle is rotatably attached through a via hole made in the bottom part of the inclined wall.
Further, the method using the cultivation apparatus, wherein the clear material is any one selected from an acrylic resin, polycarbonate, polypropylene, polyethylene, and polyvinyl chloride.
According to the above described cultivation apparatus, with a purpose of controlling a temperature of the culture solution, a water spraying member and a water spray receiving member may be installed in the outside of the cylindrical opening member arid an outer circumference of the bottom part, respectively and in addition, an artificial light source may be installed in spaces of the internal hemispheric dome, the internal conical circumferential wall, or the internal cylindrical circumferential wall.
The cultivation apparatus for algae, used in the present invention, is the cultivation apparatus for algae described in an international patent application (PCT/JP99/01585) and is that described below.
The apparatus used for cultivation according to the present invention will be described below.
The cultivation apparatus for algae, used in the present invention, is the cultivation apparatus with the dome shape selected from those with any one shape of the dome shape, conical shape, or cylindrical shape, wherein the cultivation apparatus with the dome shape comprises the external hemispheric dome made from the clear material, the internal hemispheric dome made from the clear material, and the bottom part connecting bottom end parts of both the domes, and the cylindrical opening member is installed on the top part of the external hemispheric dome and the member for leading air and/or carbon dioxide gas and the discharging member for the culture solution are installed on the bottom part, and in necessary occasions, the water spraying member and the water spray receiving member are installed in the outside of the cylindrical opening member and the outer circumference of the bottom part, respectively;
the cultivation apparatus with the conical shape is the cultivation apparatus comprising the external conical circumferential wall made of the clear material, the internal conical circumferential wall made of the clear material, and the bottom part connecting bottom end parts of both the circumferential wall, and the cylindrical opening member is installed on the top part of the external conical circumferential wall and the member for leading air and/or carbon dioxide gas and the discharging member for the culture solution are installed on the bottom part; and in necessary occasions, the water spraying member and the water spray receiving member are installed in the outside of the cylindrical opening member and the outer circumference of the bottom part, respectively;
or, the cultivation apparatus with the cylindrical shape is the cultivation apparatus comprising the external cylindrical circumferential wall having the upper wall made of the clear material, the internal cylindrical circumferential wall having the upper wall made of the clear material, and the bottom part connecting bottom end parts of both the circumferential wall, and the cylindrical opening member is installed on the central part of the upper wall of the external cylindrical circumferential wall and the member for leading air and/or carbon dioxide gas and the discharging member for the culture solution are installed on the bottom part, the member for leading air and/or carbon dioxide gas and the discharging member for the culture solution are installed on the bottom part, and in necessary occasions, the water spraying member and the water spray receiving member are installed in the outside of the cylindrical opening member and the outer circumference of the bottom part, respectively.
The gas discharging apparatus used for the cultivation apparatus for algae is the apparatus configured by opposite two square base boards, the bubble leading member, of which section has the square shape lacking the side or the reversed U-shape, opened downward, and the discharge nozzle, the bubble leading member is installed with inclination to the upper side face of the square base board, forms the upper wall made by extending the inclined wall of the upper face by bending almost horizontally in the upper end part, and has the side wall extending from both the side ends of the inclined wall and the upper wall, and each of the bottom end parts of both the side walls are jointed on the upper side face of the square base board, and
the discharge nozzle is rotatably attached through the via hole made in the bottom part of the inclined wall, wherein in necessary occasions, at least any one of the opposite squared base boards is bent to a same direction in a front end part and/or a rear end part, and weight-adjusting means is installed in at least any one of the opposite squared base boards.
The present invention is applied to cultivation by using the cultivation apparatus in combination of the above described cultivation apparatus with the gas discharge apparatus.
For the clear material used for the cultivation apparatus, any material which is clear and excellent in light transmission performance and has weather resistance and ultraviolet resistance, can be used and is exemplified by materials such as acrylic resin, polycarbonate, polypropylene, polyethylene, polyvinyl chloride, and glass. In consideration of processibility, a synthetic resin is preferable; particularly acrylic resin having the above described characteristics is the most preferable material.
In this case, an internal material and an external material can be composed of a same clear material. Different clear materials such as the acrylic resin and polyvinyl chloride can be used for constituting the external hemispheric dome and the internal hemispheric dome, respectively. Or, Different materials may be used for layering.
Gas to be led to the cultivation apparatus must contain carbon dioxide as a component and may be that of which carbon dioxide concentration has been increased by mixing carbon dioxide with air and air and carbon dioxide may be independently led to the apparatus. In this case, gas is led to the culture solution by using a leading member or the gas discharge apparatus, and in occasion, both of these.
Most preferably, carbon dioxide is used by mixing with air. When air mixed with carbon dioxide lifts up stirring the culture solution, carbon dioxide is dispersed in the culture solution to be absorbed and air works to remove oxygen generated by cultivation from the culture solution. And, when carbon dioxide is independently led to the culture solution, a leading rate becomes low to cause inevitably a delaying tendency of a dispersion rate of carbon dioxide in the culture solution.
The cylindrical opening member has an action to disperse air injected to the culture solution, carbon dioxide gas unused, and oxygen generated to atmosphere. In the opening member opened, impurities such as waste are easy to invade. In order to prevent contamination of such matter, it is preferable to install a filter member in the opening member or the lid member is installed in the opening member to give the same action as that of the filter member.
This opening member may be formed integrally with the external hemispheric dome, the external conical circumferential wall, or the upper wall of the external cylindrical circumferential wall and may be that made by fixing that which is formed as a separate body.
The dome-shaped, conical shaped, or cylindrical shaped cultivation apparatus used as the main body of the cultivation apparatus may be integrally formed by either of the external member or the internal member, may be that of which one is integrally formed and the other is assembled with members properly divided into two or four pieces, and may be that assembled with members made by dividing both the members. Construction of the apparatus may be determined on the basis of a size and the shape of the cultivation apparatus.
In addition, for the water spray receiving member, if it is the member capable of receiving a water stream falling down on an external surface of the apparatus by spraying, the material and structure thereof are free. The material is exemplified by a metal material and a plastic material.
A structure of the water spray receiving member may be that formed as a body separated from the main body of the cultivation apparatus, that in which the receiver is constructed by extending the bottom end part of the external side member of the cultivation apparatus in a horizontal direction of the outer circumference and the front end part thereof is bent upward, and that the water spray receiver is constructed by extending the bottom end part of the internal side member of the cultivation apparatus in the horizontal direction of the outer circumference and the front end part thereof is bent upward.
It is preferable to constitute the water spray receiver with the member formed as the body separated from the main body of the cultivation apparatus.
As the member, for leading air and/or carbon dioxide gas, installed in the bottom part, a tubular member, in which a plurality of gas discharge pores are prepared, may be used and that of which bottom part has gas discharge pores in the bottom part may be used. It is not necessary to stir the culture solution mechanically by gas led from this leading member to the culture solution, because the culture solution is stirred by lifting of gas climbing up in the culture solution. Therefore, according to this method, break of algal cells by mechanical stirring or generation of the shear stress can be prevented.
Further, in accordance with lifting of gas, oxygen gas generated by photosynthesis can be efficiently and rapidly discharged from the culture solution.
Methods to supply the culture solution to the cultivation apparatus are divided in two categories. The one is the method by which a supply member (for example, a supply hole made on the bottom part) is installed in the bottom part to supply the culture solution through this supply member.
The second method is the method by which the culture solution is supplied from the cylindrical opening member of the top part. Installing various supply members and leading members in the apparatus causes complexity of the apparatus and also in case changing a species of microalgae to cultivate, contamination comes up as a problem.
Hence, the second method is more preferable one.
Both the external member and the internal member of the cultivation apparatus are composed of the clear material and thus, the artificial light source installed in the internal space of the cultivation apparatus allows cultivation in night for outdoor cultivation. In addition, in the indoor cultivation, an internal and external two artificial light sources of the cultivation apparatus allow realizing efficient continuous cultivation.
The dome-shaped cultivation apparatus occupies a small area but has a large surface area and hence, has the large light-receiving area. And, in this apparatus, stirring the culture solution is very preferably operated. On the other hand, this apparatus, in case forming with a plastic resin, it is molded readily by vacuum molding to allow preparation at the lowest cost.
From these reasons, the dome-shaped cultivation apparatus is the most preferable as the cultivation apparatus for algae.
In order to control and monitor cultivation conditions, it is preferable to install various sensors such as a temperature sensor, a liquid level sensor, a pH sensor, and a dissolved oxygen amount sensor in the cultivation apparatus. These sensors are installed through the cylindrical opening member or an external wall of the apparatus.
The gas discharge apparatus used in the present invention discharges gas such as air toward the bottom part of the cultivation apparatus in an obliquely downward direction and thus, proceeds jumping like a frog in the apparatus. By this action, the culture solution is vigorously stirred and gas which is discharged lifts in the culture solution to stir the culture solution. Particularly, in the case where algae to be cultivated is easy to form a colony, gas discharged from the gas discharge apparatus breaks the colony and algal cells are dispersed in the culture solution to increase cultivation efficiency.
The gas discharge apparatus is normally composed of the plastic resin and the weight-adjusting means is installed to adjust a weight of the apparatus.
Cultivation of algae by using such cultivation apparatus gives the following advantages: 1) no contamination of bacteria and exogenous matters, 2) easy regulation of the temperature of the culture solution, 3) no break of algal cells and no occurrence of shear stress due to capability of stirring of the liquid without mechanical stirring of the culture solution, 4) capability of increase in culture density, 5) easy cleaning of the apparatus, 6) no inhibition of cultivation by oxygen generated, and 7) a high light use efficiency. In addition, in case using the gas discharge apparatus in the culture solution, necessary gas is supplied to the culture solution by using the apparatus and thus the following advantages appear: the apparatus operates a moving motion to stir the liquid and also gas discharged stirs the liquid to make contact of gas supplied with the culture solution very good resulting in increase in cultivation efficiency.